1. Field of the Invention
The present invention relates to methods and compounds for rapid identification of clusters of epidemiologically related individual strains of Mycobacterium tuberculosis by a DNA amplification strategy of multiple copy elements of M. tuberculosis. Additionally provided is a method for detecting the presence of M. tuberculosis RFLP-type 021-2072 (or strain W).
2. Background Art
Tuberculosis remains a major source of morbidity and mortality throughout the world and is increasing in the United States today. The resurgence of tuberculosis in the United States is largely related to the human immunodeficiency virus (HIV) epidemic. Because of the reduction in cell-mediated immunity in HIV-infected persons, active disease may develop quickly after exposure to Mycobacterium tuberculosis (Barnes et al., 1991). As the number of hospitalized patients infected with HIV and tuberculosis increases, the risk of nosocomial infection with M. tuberculosis increases not only among patients, but also among health care providers (Pearson et al., 1992).
An important factor in the control of tuberculosis is the ability to identify outbreaks and track the transmission of a particular strain of M. tuberculosis. The standard procedure for distinguishing strains of M. tuberculosis isolates has been phage typing; however, a more sensitive molecular approach has been described recently (Cave et al., 1991). This approach takes advantage of the facts that M. tuberculosis strains carry multiple copies of an insertion sequence, IS6110, and that the precise locations of the IS6110 elements in the M. tuberculosis genome varies significantly from strain to strain, providing a unique DNA fingerprint for each M. tuberculosis strain. The IS6110-restriction fragment length polymorphism (RFLP) technique has been shown to be a reliable and reproducible method for differentiating M. tuberculosis strains (Cave et al., 1991; Otal et al., 1991; van Soolingen et al.), and a recent report utilized this technique to study the epidemiology of multidrug-resistant tuberculosis among hospitalized HIV-infected patients (Edlin et al., 1992). This method allows for the grouping of isolates into fingerprint types.
The IS6110-RFLP procedure requires growth of the organism followed by purification of genomic DNA from the bacteria. The purified DNA is digested with a restriction enzyme which cleaves within the IS6110 sequence. The digested genomic DNA is then electrophoresed on agarose gels, transferred to a membrane, and hybridized with a portion of the IS6110 sequence. This is a time-consuming method that is useful for retrospective epidemiology but has limitations concerning disease management or a rapid response to outbreak situations because of the relatively long time period required to obtain results.
Furthermore, during 1991, the CDC investigated an outbreak of multidrug-resistant tuberculosis (MDR-TB) at a state correctional facility in New York. Eight persons (seven inmates and one correctional facility worker) were identified as having MDR-TB which were resistant to isoniazid, rifampin, ethambutol, streptomycin, kanamycin, ethionamide and rifabutin. All eight patients died within 42 days from the date of collection of their first positive sputum. Restriction fragment length polymorphism (RFLP) analysis for typing Mycobacterium tuberculosis gave identical patterns for seven of the eight strains (eighth strain pending). Epidemiologists state two factors which contributed to this outbreak are 1) length of time required to identify and isolate inmates with active TB from the general prison population and 2) length of time required for identification of M. tuberculosis and the performance of drug susceptibility tests (Greifinger et al.).
This outbreak illustrates the need for a rapid method to detect multidrug-resistant strains of M. tuberculosis including this strain designated M. tuberculosis RFLP-type 021-2072 (or strain W). The strain has increased in prevalence in the New York City area since 1991 due to its multidrug-resistance and is becoming a public health problem. Additionally, there is a strong need for a general method for rapidly, safely and accurately identifying individual strains of M. tuberculosis. In particular, a method that eliminates the need for culturing the slow-growing, highly infective virulent M. tuberculosis cells would be much more useful and desireable than any presently-existing method.